Overexpression and purification of recombinant proteins are of significant interest to the pharmaceutical and biochemical industries. Recombinant proteins, for example, are used in a variety of commercially important applications, including therapeutics, bioinsecticides, diagnostic kits and many others. Advances in recombinant deoxyribonucleic acid (DNA) technology and protein expression systems have rendered practical the production of proteins in significant quantities employing a variety of hosts. However, rapid and efficient purification of recombinant proteins remains a major challenge. Downstream purification of recombinant proteins can account for about 80% of total production cost. Therefore, cost-effective purification methods are of critical importance to pharmaceutical and biotechnology companies.
In this context, affinity chromatography is the method of choice for protein purification. This method involves addition of a selective affinity tag sequence to the target protein gene to generate the fusion gene. The fusion protein, produced by overexpression of the fusion gene in heterologous hosts, is purified by exploiting the highly specific binding characteristics of the affinity tag. The affinity tag is subsequently removed from the target recombinant protein. Several affinity tags such as polyhistidine, glutathione S-transferase, maltose binding protein, chitin, thioredoxin, small ubiquitin modifier protein (SUMO), N-utilization substance A (Nus A) and others have been popularly employed for recombinant protein purification. Nevertheless, common problems afflict these affinity tags, including decreased column capacity due to large molecular size of the affinity tags, high cost of elution solutes, tendency of the affinity tag(s) to be expressed as insoluble inclusion bodies, problems in recovery of cleaved affinity tags and difficulties in accurately maintaining the pH of solution used for protein elution.